Method for preparation of orally administrated insulin formulation

ABSTRACT

The invention relates to the method for preparation of orally administrated insulin oil formulation comprising: dissolving an amount of insulin in an acidic aqueous buffer(A); adding A to liquid surfactant, the HLB value of which is between 10 and 20, with agitation to form a homogeneous solution(B); adding B to an oil, the HLB value of which is between 0 and 10, with agitation to form oil formulation. This invention has a simple process with low cost. After orally administrated, the formulation prepared with the process of the invention can resist the gastrointestinal degradation by digestive enzymes, and is easily absorbed for hypoglycemic effect.

FIELD OF THE INVENTION

The present invention relates to the preparation of biochemical drug, inparticularly relates to the new process for preparation of orallyadministrated peptide or protein drug.

BACKGROUND OF THE INVENTION

Many biochemical drugs, such as insulin, growth hormone, calcitonin,thrombolytic enzyme can only be administrated by injection. The patientshave to suffer from the pain and discommodiousness for daily injection,even 34 injections per day of the drug like insulin, so it is desired todevelop oral route of these drugs. Great benefit of economic andsocietal would be arisen for the breakthrough in this study. Asestimated by “New Scientists” of America, the economic benefit broughtwith the success of this study could be up to six billion dollars peryear. However, due to the intestinal proteolytic degradation and thepoor penetration into the blood stream, the bioavailabilities of thesebiochemical drugs after direct orally administrated are lower than 0.5percent. To overcome the disadvantages, liposome is used in some studiesto enwrap insulin for the resistant of intestinal proteolyticdegradation, but it's absorptivity is too low to achieve approvingpharmacological effect. The insulin nanoparticles made of a-polyalkylcyanoacrylate are also be studied, but the experimentindicated that most of insulin are on the surface of nanoparticlesinstead of in the inside of hydrophobic capsule as anticipant.Therefore, although this nanoparticles can resist the intestinalproteolytic degradation in a certain extent, due to individualdifferences, it can not be used as a viable drug formulation. It is alsostudied to dissolve insulin in a hydrophobic solution or an oil phasefor oral administration, but the process is too complex. GB patent (WO95/13795, WO 97/34581) related to insulin oil formulation, it is neededto remove the hydrophilic solvent by circumrotate evaporation, sprayevaporation even lyophilisation more than two days under a temperatureof −40° C. or less and an air pressure of 0.1 millibar or less. Suchtechnical requirements restrict the industrial scale of said process andincrease the cost.

SUMMARY OF THE INVENTION

The object of the present invention is to overcome deficiency of theprior art, in particularly provides a method for preparation of orallyadministrated insulin oil formulation, which is simple and manageable aswell as with low cost. After orally administrated, the formulationprepared with the process of the invention can resist thegastrointestinal degradation by digestive enzymes, and is easilyabsorbed, thus obtaining favorable hypoglycemic effect.

The invention provides a method for preparation of orally administratedinsulin oil formulation which comprising:

1) dissolving an amount of insulin in an aqueous buffer, resulting insolution(A), of which the pH value is between 3 and 5, and in which theconcentration of insulin is 0.1–0.5 mg/mL;

2) adding acidic solution A to a liquid nonionic surfactant or anamphiphilic ester or a mixture thereof with 10^HLB (Hydrophile-LipophileBalance)<20, wherein the volume ratio of solution A to that of thesurfactant or the amphiphilic ester or their mixture is from 1:5 to1:50, and then mixing at 5–30° C. with agitation to form homogeneoussolution (B);

3) adding solution B to an hydrophobic emulsifier or the mixture thereofwith 0<HLB<10, wherein the volume ratio of solution B to that of ahydrophobic emulsifier or their mixture is from 1:1 to 1:10, mixing thistwo solution at 5–30° C. with agitation to form an oil formulation(C),which is stored at 4–10′C;

4) adding an amount of antioxidant, wherein when the antioxidant ishydrophobic, the antioxidant is added to solution B during step 3), andwhen the antioxidant is hydrophilic, the antioxidant is added tosolution A during step 2). The surfactant or amphiphilic ester or theirmixture mentioned in step 2) may be one or more selected from the groupconsisting of decaglycerol monooleate (HLB=12.9), hexaglycerolmonolaurate (HLB=13.5), decaglycerol monooctanoate (HLB=16),polyethylene glycol-8-glycerol octanoate/decanoate: (HLB=14),polyglycerol-6-dioleate (HLB=10), Tween 80 (sorbitan monooleatepolyoxyalkylene) (HLB=15.4), phospholipids, glycoester, bile acid andits salt, and other biological surfactant. The agitation speed in step2) may be 200–2000 r/min, and the agitation time is in the range of0.5–5 hours.

The oil or hydrophobic emulsifier or their mixture mentioned in step 3)may be one or more selected from the group consisting of triglyceroloctanoate/decanoate (HLB=1), glycerol octanoate/decanoate (HLB=3),glycerol oleate (HLB=3), glycerol linoleate (HLB=3),polyglycerol-3-oleate (HLB=6), polyethylene glycol-6-glycerol monooleate(HLB=4), polyethylene glycol-6-glycerol linoleate (HLB=4), Span 80(sorbitan monooleate) (HLB=4.3), polyethylene glycol-4-glyceroloctanoate/decanoate (HLB=5). The agitation temperature in step 3) is inthe range of 5–30° C., the agitation speed is in the range of 200–2000r/min, and the agitation time is in the range of 0.5–5 hours.

The amount of the antioxidant added may be 0.01–0.1% (volume ratio)relative to that of the final solution. The examples of the antioxidantare Vitamin E, Vitamin C, cysteine, gallate, tertiarybutyl-hydroquinone.

The obtained oil formulation can be orally administrated directly, orenveloped into capsule (soft capsule or capsule with liquid inclusion),and can also be mixed with some kind of pharmaceutically acceptablesolid excipients such as amylum, dextrine, ethylane cellulose,monostearate, in order to form troche or capsule with solid particleinclusion.

The present invention can also be used in preparation of other peptideor protein oil formulation.

The method of the invention is simple and manageable and with low cost.The dispersants and oils used in the invention are low toxicity andsafety for oral administration. Insulin is even dissolved in oil phaseto form a transparent insulin oil formulation. It was indicated byexperiment in vitro that the formulation prepared with the process ofthe invention could be emulsified in the solution the pH value of whichcould be from 2 to 11, and the insulin are still in the oil phaseinstead of into the aqueous phase. After orally administrated, thus, theformulation prepared with the process of the invention can resist thegastrointestinal degradation by digestive enzymes, and is easilyabsorbed, thus obtaining favorable hypoglycemic effect.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is the curve of hypoglycemic effect in diabetic rats after orallyadministrated the insulin oil formulation prepared by the process of theinvention.

EXAMPLES Example 1

1. 0.5 g of insulin was dissolved in 5 ml of buffer with the pH value of4, resulting in a solution (A).

2. 2 ml of Tween 80 and 23 ml of decaglycerol monooctanoate were mixedtotally, 15 then solution A was added under agitation with the agitationspeed of 800 r/min, the agitation period of 2 hours and the temperatureof 20° C., obtaining in solution (B).

3. Solution B was added to 70 mL of glycerol oleate under agitation toform solution(C) of 100 ml total volume with the agitation speed of 800r/min, the period of 3 hours and the temperature of 20° C.

4.60 u l of propyl gallate was dissolved in solution C, then stored inthe icebox for use.

Example 2

1. 1 g of insulin and 50 mg of Vitamin C were added to 5 ml of bufferwith the pH 25 value of 4 for dissolving, and obtaining solution A.

2. Solution A was added to 40 ml of polyethylene glycol-8-glyceroloctanoate/decanoate under agitation to form solution (B) of 45 ml totalvolume with the agitation speed of 1500 r/min, the agitation period of 2hours and the temperature of 24″C. 30

3. 27.5 ml of polyglycerol-3-oleate and 27.5 ml of polyethyleneglycol-6-glycerol monooleate were mixed, then solution B was added underagitation with an agitation speed of 1500 r/min, the agitation period of2 hours and the temperature of 24° C. to form a solution (C), which wasstored in the icebox for use.

Example 3

1. 0.8g of insulin was dissolved in 4 ml of buffer with the pH value of4, resulting in a solution (A).

2. Solution A was added to 20 mL of polyethylene glycol-8-glyceroloctanoate/decanoate to form a solution (B) of 24 mL total volume underagitation with the agitation speed of 500 r/min, the agitation period of2.5 and the temperature of 18° C.

3. Solution B was added to 76 mL of polyglycerol-3-oleate to form asolution(C) of 100 ml total volume under agitation with the agitationspeed of 500 r/min, the agitation period of 3 hours and the temperatureof 18° C.

4. 80 n l of vitamin E was added to solution C, the resulting mixturewas mixed totally, then stored in the icebox for use.

Animal experiments were done for the insulin oil formulation prepared bythe process of the examples. Hypoglycemic effect in diabetic rats (n=10)after orally administrated the insulin oil formulation (25 IU/kg) wasshowed as FIG. 1.

1. A method for preparation of orally administrated insulin formulation,comprising: 1) dissolving an amount of insulin in an aqueous buffer,resulting in solution A, of which the pH value is between 3 and 5, andin which the concentration of insulin is 0.1–0.5 mg/mL; 2) adding acidicsolution A to a liquid nonionic surfactant or an amphiphilic ester or amixture thereof with 10<HLB<20, wherein the volume ratio of solution Ato that of the surfactant or the amphiphilic ester or their mixture isfrom 1:5 to 1:50, and then mixing at 5–30° C. with agitation to formhomogeneous solution B; 3) adding solution B to an hydrophobicemulsifier with 0<HLB<10, wherein the volume ratio of solution B to thatof the hydrophobic emulsifier is from 1:1 to 1:10, and then mixing at5–30° C. with agitation to form formulation C, which is stored at 4–10°C.; 4) adding an amount of antioxidant, wherein when the antioxidant ishydrophobic, the antioxidant is added to solution B during step 3), andwhen the antioxidant is hydrophilic, the antioxidant is added tosolution A during step 2); wherein the formulation does not include anoil.
 2. The method of claim 1, wherein the surfactant or amphiphilicester or their mixture mentioned in step 2) is one or more selected fromthe group consisting of decaglycerol monooleate, hexaglycerolmonolaurate, decaglycerol monooctanoate, polyethylene glycol-8-glyceroloctanoate/decanoate, polyglycerol-6-dioleate, sorbitan monooleatepolyoxyalkylene (20), phospholipids, glycoester and bile acid or itssalt as biological surfactant.
 3. The method of claim 1, wherein theagitation speed in step 2) is in the range of 200–2000 r/min, and theagitation time is in the range of 0.5–5 hours.
 4. The method of claim 1,wherein the hydrophobic emulsifier mentioned in step 3) is one or moreselected from the group consisting of triglycerol octanoate/decanoate,glycerol octanoate/decanoate, glycerol oleate, glycerol linoleate,polyglycerol-3-oleate, polyethylene glycol-6-glycerol monooleate,polyethylene glycol-6-glycerol linoleate, sorbitan monooleate, andpolyethylene glycol-4-glycerol octanoate/decanoate.
 5. The method ofclaim 1, wherein the agitation temperature in step 3) is in the range of5–30° C., the agitation speed is in the range of 200–2000 r/min and theagitation time is in the range of 0.5–5 hours.
 6. The method of claim 1,wherein the amount of the antioxidant added is in the range of 0.01–0.1%by volume of the final solution, and said antioxidant comprises VitaminE, Vitamin C, cysteine, gallate, or tertiary-butyl hydroquinone.